TREATMENT OF MYCOBACTERIAL DISEASES BY ADMINISTRATION OF BACTERICIDAL/PERMEABILITY-INCREASING PROTEIN PRODUCTS

Abstract: The present invention relates to methods for treating a subject suffering from infection with Mycobacteria, such as M. leprae or M. tuberculosis comprising administering to the subject a composition comprising a bactericidal/permeability-inducing (BPI) protein product.

Description: Treatment of Mycobacterial Diseases by Administration of Bactericidal/Permeability-Increasing Protein Products Background of the Invention The present invention relates to methods of treating a subject suffering from infection with Mycobacteria by administration of Bactericidal/Permeability-Increasing Protein (BPI) protein products. Mycobacterium is a non-motile, acid-fast, aerobic, Gram-positive genus of bacteria known to cause grave human and animal diseases, such as tuberculosis and leprosy. Infections caused by M.avium are the most common form of disseminated bacterial disease in AIDS patients. Orme, et al. , Infect, and Immun., 67^:338-342 (1993). The administration of conventional antibiotics to treat Mycobacterial infection is known in the art and has achieved varying success depending on the susceptibility of the bacterial strain, the efficacy and toxicity of the antibiotic(s) employed, the duration of treatment, and numerous other factors. Antimicrobials that have been employed alone or in combination in the United States to treat tuberculosis include isoniazid, rifampin, ethambutol, 7-aminosalicylic acid, pyrazinamide, streptomycin, capreomycin, cycloserine, ethionamide, and kanamycin. McClatchy, Antimycobacterial Drugs: Mechanisms of Action, Drug Resistance, Susceptibility Testing, and Assays of Activity in Biological Fluids, pp. 134-197, In Antibiotics in Laboratory Medicine, 3rd ed., V. Lorian, ed. The Williams & Wilkins Co., Baltimore (1991). As many Mycobacterial strains are drug resistant, serious obstacles exist for control and successful treatment of tuberculosis and other Mycobacterial diseases. Id. A variety of factors have made treatment of individuals afflicted with Mycobacterial diseases problematic. First, Mycobacteria possess a very hydrophobic cell wall that affords protection against the host's immune system. As Mycobacterial infections tend to be chronic, the pathologies of these organisms are generally due to host response. Also, many Mycobacterial strains are drug-resistant. These and other factors make the development of novel, effective methods for treating Mycobacterial diseases highly desirable. Mycobacteria are readily distinguished from Gram-negative bacteria by selective staining (i.e., Acid-fast or Gram's stain) due to significant differences in cell wall structure. Gram-negative bacteria are characterized by a cell wall composed of a thin layer of peptidoglycan covered by an outer membrane of lipoprotein and lipopolysaccharide (LPS), whereas Gram-positive bacteria have a cell wall with a thicker layer of peptidoglycan with attached teichoic acids, but no LPS. One of the major constituents widely distributed within the cell wall of Mycobacterium species is lipoarabinomannan (LAM). LAM has been purified from both M. leprae and M. tuberculosis. Hunter et al., J Biol. Chem., 267:12345-12351 (1986). LAM is a serologically active mannose containing phosphorylated lipopolysaccharide that may be membrane associated. The complex physiological effects of LAM appear to be concentration, time, and source-dependent. For example, Chaterjee et al. , Infect, and Immun., 60(3,1:1249-1253 (1992), reported that, in the first 24 hours following exposure, LAM from an avirulent strain of tuberculosis was 100-fold more potent at stimulating TNF secretion in mouse macrophages than LAM from a virulent strain. LAM concentrations of 0.01-10 μg/πύ. for the avirulent strain and 0.01-100 g/ml for the virulent strain were tested, and increased LAM concentration was associated with increased TNF production with LAM from both species. Macrophage-inhibitory effects of LAM have also been described in the art. LAM purified from both M. leprae and M. tuberculosis has been reported to be a potent in vitro inhibitor of T-cell lymphokine activation of mouse macrophages. Sibley et al. , Infection and Immunity, 56(5):1232-1236 (1988). Because the principle efferent role of the macrophage in acquired resistance to inrracellular pathogens requires activation by T-cell lymphokines, notably gamma-interferon (EFN-7), macrophages whose activation-response is inhibited are severely compromised in their capacity for both enhanced microbicidal and tumoricidal activities. In another study, Sibley et al. , Clin. Exp. Immunol. , 80(7,1:141- 148 (1990), reported that pretreatment of mouse macrophages with 50 to 100 ug/ml LAM blocked macrophage activation by IFN-γ, but pretreatment with 10 μg/ml LAM did not affect macrophage activation. Thus, it is believed that low concentrations of LAM stimulate cytokine production, at least initially. However, higher concentrations of LAM (50-100 μglmi or more) appear to block rather than promote macrophage function. Thus, the production of either too much or too little cytokine at different stages of Mycobacterial disease may contribute to Mycobacterial pathogenesis. New methods for blocking the above-characterized physiological effects of LAM molecules are a highly desirable goal in the treatment of subjects that are or that have been infected with Mycobacteria. For the same reasons, new methods by which fluids containing LAM can be decontaminated prior to administration into a subject are also desirable. Neutralization of even small amounts of LAM is desirable, because small amounts of LAM may have the physiological effect of stimulating cytokine production. Of interest to the background of the invention are the disclosures of PCT/US88/00510, (WO 88/06038) published August 25, 1988, indicating that certain poloxypropylene/polyoxyethylene nonionic surface-active block copolymers can be used with or without conventional antibiotics to treat infection with Mycobacterium. This reference cites studies suggesting that the effects of other nonionic surfactants on tuberculosis are most likely due to modification of surface lipids of Mycobacteria, and not to direct bactericidal effects on Mycobacteria. See e.g. Cornforth et al. , Nature, 768:150-153 (1951). Bactericidal/permeability-increasing protein (BPI) is a protein isolated from the granules of mammalian polymorphonuclear neutrophils (PMN), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMN's by acid extraction combined with either ion exchange chromatography Elsbach, 7. Biol. Chem., 254:11000 (1979) or E. coli affinity chromatography, Weiss, et al., Blood, 69: 652 (1987), and has potent bactericidal activity against a broad spectrum of Gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 Daltons (55 kD). The amino acid sequence of the entire human BPI protein, as well as the DNA encoding the protein, have been elucidated in Figure 1 of Gray, et al. , J. Biol. Chem., 264: 9505 (1989), incorporated herein by reference. BPI has been shown to be a potent bactericidal agent active against a broad range of Gram-negative bacterial species. The cytotoxic effect of BPI has been shown to be highly specific to sensitive Gram-negative species, while non-toxic for other non-acid fast, Gram-positive bacteria and for eukaryotic cells. The precise mechanism by which BPI kills bacteria is as yet unknown, but it is known that BPI must first attach to the surface of susceptible Gram-negative bacteria. It is thought that this initial binding of BPI to the bacteria involves electrostatic interactions between the basic BPI protein and negatively charged sites on lipopolysaccharides (LPS). LPS has been referred to as endotoxin because of the potent inflammatory response that it stimulates. LPS induces the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to Lipid A, the most toxic and most biologically active component of LPS. In susceptible bacteria, it is thought that BPI binding disrupts LPS structure, leads to an activation of bacterial enzymes that degrade phospholipids and peptidoglycans, alters the permeability of the cell's outer membrane, and ultimately causes cell death by an as yet unknown mechanism. BPI is also capable of neutralizing the endotoxic properties of LPS to which it binds. Because of its Gram-negative bactericidal properties and its ability to neutralize LPS, BPI can be utilized for the treatment of mammals suffering from diseases caused by Gram-negative bacteria, such as bacteremia or sepsis. An approximately 25 kD proteolytic fragment corresponding to the ammo-terminal portion of human BPI holoprotein possesses the antibacterial efficacy of the naturally-derived 55 kD human holoprotein. In contrast to the amino-terminal portion the carboxy-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity. Ooi, et al, J. Exp. Med., 174:649 (1991). A BPI amino-terminal fragment, comprising approximately the first 199 amino acid residues of the human BPI holoprotein, has been produced by recombinant means as a 23 kD protein referred to as "rBP^". Gazzano-Santoro et al, Infect. Immun. 60: 4754-4761 (1992). Of interest to the present invention is the disclosure of Kelly et al. at the Annual Meeting of the Society of University Surgeons, Feb 10-14, 1993, (paper #1) that intravenously administered BPI is useful for providing improved pulmonary clearance in a Gram-negative pneumonia model. While BPI protein products are effective for treatment of conditions associated with Gram-negative bacterial infection, there continues to exist a need in the art for products and methods for treatment of Gram-positive bacterial infections such as infection with Mycobacteria. SUMMARY OF THE INVENTION The present invention provides methods of treating a subject suffering from infection with Mycobacteria by administration of a composition comprising a BPI protein product. Therapeutic compositions according to the invention may be administered orally, systemically (such as by intravenous, intramuscular or other injection), or as an aerosol. Mycobacterial disease states subject to treatment according to the invention include tuberculosis, which can be caused by infection with M. tuberculosis, leprosy, which can be caused by infection with M. leprae, and diseases caused by M.avium and other Mycobacteria species. According to preferred methods, antibiotics and/or surfactants may be administered in combination with the BPI protein product to subjects suffering from infection with Mycobacteria. According to another aspect of the present invention, compositions comprising a BPI protein product are administered to neutralize LAM's physiological effects on a host. For example, methods are provided for neutralizing the effect of low concentrations of LAM capable of stimulating cytokine production in a host. Methods are also provided for neutralizing the inhibitory effect that higher concentrations of Mycobacterial LAM (i.e. 100 μg/ml or more) have upon the interferon-mediated activation of macrophages. Specifically, a BPI protein product may be administered to an immunosuppressed subject failing to respond to microbes or tumor cells due to LAM-induced insensitivity of macrophages to activation by T-cell lymphokines. According to a further aspect of the present invention, a BPI protein product is employed in methods for decontaminating a fluid containing LAM prior to administration of the fluid into a subject. Such decontamination methods of the invention involve contacting the fluid with the BPI protein product prior to administration, under conditions such that LAM forms a complex with the BPI protein product which can be removed from the fluid. Fluids subject to decontamination by the methods of this invention include, but are not limited to, blood, plasma, blood serum, bone marrow, isotonic solutions, pharmaceutical agents, and cell culture agents. A further aspect of this invention relates to the use of a composition comprising a BPI protein product for the manufacture of a medicament for the therapeutic application of treating any of the aforementioned conditions or infections from which a subject might suffer. As used herein, "BPI protein product" includes naturally and recombinantly produced bactericidal/permeability-increasing protein, natural, synthetic and recombinant biologically active polypeptide fragments of bactericidal/permeability-increasing protein, and biologically active polypeptide analogs, including hybrid fusion proteins, of either bactericidal/permeability- Increasing protein or biologically active fragments thereof. Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon considering the following detailed description of the invention, which describes presently preferred embodiments thereof. BRIEF DESCRIPTION OF THE FIGURES Fig. 1 graphically depicts the results of an assay of BPI protein product binding to E. coli J5 Lipid A and M. tuberculosis. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the discovery that a composition comprising a BPI protein product can be administered for effective treatment of a subject suffering from infection with Mycobacteria. In particular, the invention provides methods for treatment of leprosy and tuberculosis, grave diseases caused by the species M. leprae and M. tuberculosis, respectively. It is contemplated that the methods described herein may be used to treat infection with other Mycobacterial species, most notably M. avium, but also M. marinum, M. fortuitum, M. chelonei, and the like. Beneficial effects of treatment with BPI protein products are expected to result from binding of the products to LAM and to disruption of the bacterial cell wall components (with or without direct killing of the bacteria) in manner similar to that resulting from treatment of Gram-negative disease states. The BPI protein products including biologically active fragments of BPI holoprotein which are to be administered according to the methods of this invention may be generated and/or isolated by any means known in the art. Co-owned, copending U.S. Patent Application Ser. No. 07/885,501, hereby incorporated by reference herein, discloses novel methods for the purification of recombinant BPI protein products expressed in and secreted from genetically transformed mammalian host cells in culture and how one may produce large quantities of recombinant BPI protein products suitable for incorporation into stable, homogeneous pharmaceutical preparations. Biologically active fragments of BPI include biologically active molecules that contain the same amino acid sequence as a BPI holoprotein, except that the molecules lack amino-terminal amino acids, internal amino acids, and/or carboxy-terminal amino acids of the holoprotein. By way of nonlimiting examples, such fragments include the previously mentioned natural 25 kD fragment and the recombinant 23 kD, 199 amino acid residue amino- terminal fragment of human BPI holoprotein referred to as rBPI- j . See, Gazzano-Santoro et al , Infect. Immun. 60:4754-4761 (1992). In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBP^ ) having the 31 -residue signal sequence and the first 199 amino acids of the amino-terminus of the mature human BPI, as set out in SEQ ID NOs: 1 and 2 taken from Gray et al, supra, except that valine at position 141 is specified by GTG rather than GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG). Biologically active fragments of BPI also include, but are not limited to those polypeptides disclosed in co-owned, co-pending, U.S. Patent Application Serial No. 08/030,644, filed March 12, 1993 by Little et al, entitled "Therapeutic Uses of Bactericidal Permeability-Increasing Protein Products" and noted to display the LPS binding, bactericidal and heparin binding activities of BPI holoprotein. Biologically active polypeptide analogs of BPI include but are not limited to biologically active hybrid fusion proteins comprising a BPI protein or biologically active fragment thereof, and at least a portion of at least one other polypeptide. Such proteins are described by Theofan et al. in co- owned, co-pending U.S. Pat. App. Ser. No. 07/885,911, which is incorporated herein by reference in its entirety, and include hybrid fusion proteins comprising, at the amino terminal end, a BPI protein or a biologically active fragment thereof and, at the carboxy terminal end, at least one constant domain of an immunoglobulin heavy chain or allelic variant thereof. Biologically active peptide analogs of BPI also include but are not limited to a BPI protein or biologically active fragment thereof, wherein one or more amino acid residues has been replaced by a different amino acid. For example, co-owned, copending U.S. Patent Application Ser. No. 08/013,801 (Theofan et al , "Stable Bactericidal/Permeability-Increasing Protein Products and Pharmaceutical Compositions Containing the Same," filed February 2, 1993, which is incorporated herein by reference, discloses polypeptide analogs of BPI and of BPI fragments wherein a cysteine residue at position 132 or at position 135 is replaced by a different amino acid. The administration of a BPI protein product is preferably accomplished with a pharmaceutical composition comprising the BPI protein product and a pharmaceutically acceptable diluent, adjuvant, or carrier. The BPI protein product composition may be administered without or in conjunction with known antibiotics, surfactants, or other chemotherapeutic agents. A preferred pharmaceutical composition comprises the BPI protein at a concentration of 1 mg/ml in citrate buffered saline (0.02 M citrate, 0J5 M NaCl, pH 5.0) comprising 0.1% by weight of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, NJ) and 0.002% by weight of polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington, DE). Such preferred combinations are taught in a co-owned, copending, U.S. Patent Application Ser. No. 08/012,360 (McGregor et al., "Improved Pharmaceutical Compositions" filed February 2, 1993); the disclosure of which is incorporated herein by reference. The surfactants which may be employed may also have the advantage of increasing the availability to BPI protein products of LAM binding sites, by removing some of the waxy rMycobacterial cell wall components; surfactants may also enhance the disassociative activity of BPI protein products on the cell membrane. Some surfactants are themselves anti- Mycobacterial and may synergize BPI protein products and vice versa. The BPI protein product can be administered by any known method, such as orally, systemically (such as by intravenous, intramuscular or other injection), or as an aerosol. Medicaments can be prepared for oral administration or by injection or other parenteral methods and preferably include conventional pharmaceutically acceptable carriers and adjuvents as would be known to those of skill in the art. The medicaments may be in the form of a unit dose in solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, and injectable and infusible solutions. Effective dosage ranges from about 100 μg/kg to about 10 mg/kg of body weight are contemplated. Intravenous administration is a preferred method for treatment of leprosy. It is contemplated that aerosol administration to the lungs will be a preferred method for treating other Mycobacterial infections, such as tuberculosis. Such aerosol formulations would be manufactured by means that are known in the art, and administered by metered-dose inhaler, updraft nebulization, or other means known in the art. An aspect of the present invention is to provide methods of treating a subject suffering from any of the physiological effects of Mycobacterial LAM. As described above, the physiological effects of LAM depend on a number of factors, including the source and concentration of the LAM, and the length of time to which host cells are exposed to LAM. Example 3, infra, demonstrates that 20 - 100 μg/ml of nonviable, desiccated M. tuberculosis added to whole blood will stimulate TNF production by the monocytes in the blood. Other studies described above have shown that 50- 100 g/ml of LAM will down-regulate macrophage functions and expression (TNF, and the like) and prevent macrophage activation, said methods comprise administering a BPI protein product to the subject. Methods are provided for treating a subject suffering from the effects of increased cytokine production caused by the physiological presence of LAM. Methods are also provided for treating a subject suffering from LAM-induced inhibition of macrophage activation, and the effects thereof. Methods and formulations by which a BPI protein product may be administered, including preferred methods and formulations, are the same as those set forth above for the treatment of Mycobacterial infection. Because of the harmful physiological effects that Mycobacterial LAM can have on a subject, even in the absence of viable Mycobacteria, methods are provided in the present invention by which a fluid containing LAM may be decontaminated prior to administration of the fluid into a subject. Such methods comprise contacting the fluid with a BPI protein product prior to administration, under conditions such that LAM forms a complex with the BPI protein product, thereby decontaminating the fluid. By way of nonlimiting examples, such methods may be applied to fluids such as blood, plasma, blood serum, bone marrow, isotonic solutions, pharmaceutical agents, or cell culture reagents. An aspect of this invention includes the use of a composition comprising a BPI protein product for the manufacture of a medicament for the therapeutic application of treating any of the aforementioned conditions or diseases from which a subject suffers. The medicament may include, in addition to a BPI protein product, other chemotherapeutic agents such as antibiotics or surfactants. The medicament may additionally or alternatively include one or more additional pharmaceutically acceptable components, such as diluents, adjuvants, or carriers. Methods of the present invention are more fully illustrated by the nonlimiting examples which follow. Example 1 address BPI protein products binding to a species of Mycobacterium, M. tuberculosis. Example 2 address prospective use of BPI protein products in binding purified LAM of Mycobacteria. Example 3 describes attempts to reverse Mycobacteria-induced cytokine production in whole human blood. Remaining Examples 4-11 address prospective in vitro and in vivo use of BPI protein products according to methods of this invention. The models described in those examples and/or other models known in the art are used to predict the efficacy and the optimal BPI protein product formulations of the methods of invention. EXAMPLE 1 An enzyme linked immunosorbent assay (ELISA) was conducted to determine binding of a BPI protein product to M. tuberculosis. Specifically, non-viable, desiccated M. tuberculosis H37 RA (Difco, Detroit MI) was suspended in DPBS (25 μg/ml) and used to coat microtiter wells overnight at 37 * C. Wells were also coated with either 25 μg/ml Lipid A (E.coli J5 mutant, RIBI, Hamilton MT) or 500 μl DPBS to demonstrate the functionality and specificity of rBPI 23 . After washing (3x with DPBS + 0.05% Tween 20), the plates were blocked for lhr. at room temperature with 200 μl/well of DPBS + 1 % non-fat milk. After washing as above, 50 μl solutions of either various concentrations of rBPI 2 3 (in DPBS containing 0.05% Tween 20) or DPBS (negative control) were added to the wells, which were then incubated for 1 hr. at 37 * C. The wells were again washed as above, and the amount of rBP^ bound to the wells was determined using an anti-rBP^ mouse monoclonal antibody (designated αBPI MAb-2-4) and an enzyme conjugated anti-murine IgG antibody (HRP-Ab, Zymed #61-0120, San Francisco, CA). To each well 100 μl of αBPI MAb-2-4 was added (100 ng/ml in DPBS + 0.05% Tween 20), and the plates were incubated 1 hr. at 37'C. After washing as above, 100 μl of HRP-Ab was added (1:1000 in DPBS + 0.05% Tween 20) to each well and the plates were again incubated 1 hr. at 37 * C. After washing the plates as above, lOOμl substrate in 0JM citrate plus 1:50 ABTS (20 mg/ml stock) and 1:1000 H2O2 was added to each well. The plates were incubated 10-30 min. at room temperature, and absorbance readings were taken at 405 nm (OD 405). The results of the experiment are represented graphically in Figure 1, which depicts the ability of varying concentrations rBP^ to bind to J5 Lipid A (filled triangles); to M. tuberculosis (open squares); and to the no antigen-free control (filled circles). The abscissa of each measurement represents the concentration of rBPJ.23, and the ordinate represents the average OD 405 measurements from four trials. Error bars reflect the variation in OD 405 readings for each data point. This experiment demonstrated that rBP^ binds specifically to non-viable desiccated M.tuberculosis. The functionality of the rBP^ used in these experiments was confirmed by the results of the Lipid A (positive control) binding assay, and the specificity of the experiments was confirmed by the lack of binding to the negative control samples. EXAMPLE 2 In this example, an ELISA Assay is conducted to determine binding of a BPI protein product to the lipoarabinomannan portion of Mycobacteria. The binding activity of BPI protein product (e.g., rBPk^) to LAM is demonstrated as described in the previous example, except LAM purified from a species of Mycobacterium, (e.g., M.tuberculosis or M.leprae) is substituted for the nonviable M.tuberculosis used to coat the ELISA plates in that example. Purified LAM is isolated as described by Hunter et al. , J. Biol. Chem. , 267: 12345-12351 (1986). Specific binding of biologically active BPI protein product is demonstrated by comparison of the OD 405 readings from the LAM coated wells with positive and negative controls. EXAMPLE 3 The following experiment was conducted to determine the effect of a BPI protein product, rBP^, on Mycobacteria-wώiced cytokine production in whole human blood. Whole human blood from healthy volunteers was collected into Nacutainer tubes (ACD, Beckton Dickinson, Rutherford, ΝJ). Aliquots of blood (225 μl) were mixed with either rBPI 2 3 (10 μg/ml final) or the protein thaumatin (10 μg/ml final in 5 ml) as a negative control. RPMI medium (20 μl) was added to each sample. Varying dilutions (0 - 8 ng/ml) of either E. coli 0113 LPS (Ribi, Hamilton MT) or of non- viable, desiccated M. tuberculosis H37 RA (0 - 100 μg/ml) (Difco, Detroit MI) were added to the samples, which were then incubated at 37 * C for 6 hours. The reactions were stopped by the addition of 750 μl of RPMI medium, the samples were centrifuged at 500 g for 7 min, and stored at -20 * C until analyzed. The supernatant was assayed for cytokine (TNF) levels based on a standard curve, according to the manufacturers' recommendation (Biokine ΕLISA test, T Cell Sciences, Cambridge, MA). The assay results revealed that rBPJ^ at 10 μg/ml had no inhibitory effect on M. tuberculosis-induced TNF release at the concentration (20 - 100 μg/ml) of M. tuberculosis added to the blood samples. The same concentration of rBP^ eliminated LPS-induced TNF release at the LPS concentrations tested (2 - 8 ng/ml). The lack of inhibitory effect on cytokine induction by M. tuberculosis may be the result of use of sub-optimal dosage levels. Alternatively, some component of the Mycobacterial cell wall other than the LAM bound by rBP^ may be responsible for inducing cytokine production at the Mycobacterium concentrations tested. EXAMPLE 4 The following experiment is conducted to determine the in vitro inhibitory effect of a BPI protein product on the growth of a Mycobacterium species, Mycobacterium tuberculosis (MTB). The procedure can be performed with other cultivable Mycobacterial species and employs concentrations of a BPI protein product that would be readily generated in human serum by ordinary modes of oral or parenteral administration and/or readily delivered to lung surface by aerosol administration. The effects of the BPI protein product can be evaluated with and without non-ionic surfactants, and/or standard antibiotics. Log phase cultures of antibiotic-sensitive and antibiotic-resistant MTB are incubated in either 7H11 broth medium or whole human blood, to which the following is added: (a) nothing; (b) surfactant; (c) standard MTB antibiotic; (d) antibiotic plus surfactant. Cultures are incubated with varying concentrations of, e.g., rBPl23- Duplicate cultures grown in each medium are also left untreated by rBP^ as a negative control. The organisms are placed in Bactec ® bottles (Johnston Laboratories, Cockeysville, MD) containing l C labeled nutrients. RBPI23 challenged M. tuberculosis growth is determined by measuring the elution of CO from the medium, compared to the appropriate negative control. The absence of the formation CO2 by the treated cultures is indicative of the inhibitory affects of rBP^ to MTB. Differential amounts of CO2 formed in the absence or presence of standard MTB antibiotics and or surfactants is indicative of the synergistic or additive effect that a BPI protein product has when used conjunctively with such agents. By comparing the results of this experiment performed with varying concentrations of the BPI protein product, the effective concentration of the BPI protein product is optimized. Radiometric assays to test the susceptibility of Mycobacterial species to drugs have been described previously. See McClatchy (cited supra) and references therein. EXAMPLE 5 The following experiment is conducted to determine the in vitro effects of a BPI protein product (rBP^) in an M. leprae model. A palmitic acid oxidation assay is used to measure the "viability" of the uncultivable leprosy bacillus adhered to filter paper and "grown" in a 14 C-palmitic acid- containing medium. In this method CO2 evolved from the metabolism by M. leprae of 4 C-palmitic acid is trapped on filter paper moistened with NaOH and radioactivity is determined with a liquid scintillation counter. Susceptibility to BPI protein product formulations is determined by differences in radioactivity for M. leprae tested with such formulations and treated control cultures. EXAMPLE 6 The following experiment, which is a variation of an assay conducted by Mittal et al , J. Clin. Microbiol , 17(4):704-707 (1983), is conducted to determine the in vitro inhibitory effect of BPI protein product on the growth of Mycobacterium leprae. The effects of different concentrations of BPI protein product on M. leprae are evaluated with and without non-ionic surfactants, and or standard antibiotics. The procedures as described by Mittal et al. are outlined below. Skin biopsy specimens from lepromatous patients are homogenized and are used to inoculate suspensions of mouse peritoneal macrophages cultured in RPMI 1640 (GIBCO Biocult, Irvine, Scotland) enriched with 30% fetal calf serum. After incubating 18 hours, fresh media containing [ etΛy/- 3 H]-thymidine (Amersham International Ltd., Arlington Heights, IL) is added and the cultures are incubated for 14 days. The procedure of Mittal et al. is varied by testing the effect of different concentrations of BPI protein product with or without surfactants and/or antibiotics on 3H-thymidine incorporation. Macrophages containing phagocytosed viable M. leprae will incorporate 3 H-thymidine at a 2 to 10-fold higher rate than control cultures containing heat killed M. leprae. Greater than 50% inhibition of -thymidine-incorporation is indicative of bactericidal efficacy of the test product. EXAMPLE 7 An experiment is conducted to determine the in vivo effect that a BPI protein product will have on M. tuberculosis species. The model employed is a variation of that used by Lalande et al, Antimicrobial Agents and Chemotherapy, 57^:407-413 (1993), to assess the efficacy of antimicrobial agents against M.tuberculosis. Mice inoculated intravenously with M.tuberculosis are treated with various BPI protein product doses alone or in combination with surfactants and/or antibiotics. The efficacy of such treatment regiments is analyzed as described. EXAMPLE 8 The following experiment is conducted to determine the effect that a BPI protein product will have on M. leprae in vivo. The model to be used is a variation of that developed by Shepard to study the effect of compounds on the growth of M. leprae in the footpads of infected mice. Shepard et al , Proc. Soc. Exp. Biol. Med. , 709:636-638 (1962); Shepard, J. Exp. Med. 772:445-454 (1960). Briefly, leprosy bacilli are inoculated into foot-pads of mice, which are subsequently treated with different amounts of test compound with or without known antibiotics and/or surfactants. Untreated infected mice are used as a control. Mice from each treatment regimen are sacrificed at monthly intervals, and sections cut from the infected foot. The presence of an area containing acid-fast bacteria can be observed microscopically and/or the number of such bacteria can be counted. See Shepard and McRae, Int. J. Lepr. , 56:78-82. Differences between M. leprae bacteria levels observed in treated versus control mice is indicative of the bacteriostatic or bactericidal efficacy of a given BPI treatment regimen. The metabolic status of isolated M. leprae may also be measured. Franzblau and Hastings, Antimicrobial Agnes and Chemotherapy, 57(5j:780-783 (1987). EXAMPLE 9 The following experiments are designed to demonstrate that BPI protein product is able to inhibit the ability of low concentration of LAM to induce cytokines, yet reverse the unresponsive state that attends higher concentrations of LAM. Increasing concentrations of LAM are pretreated with BPI protein product at varying concentrations. These complexes are applied to peritoneal macrophages from normal and Mycobacterium species infected mice. TNF production by treated cells will be assessed. EXAMPLE 10 A variation of the armadillo model developed by Kirchheimer et al , Int. J. Lepr. , 39:693-702 (1971); Id. , 40:229 (1972), is employed to study the in vivo effect of BPI protein product test compositions on the growth of M. leprae in infected armadillos. Briefly, leprosy bacilli are inoculated into armadillos, which are subsequently treated with different amounts of a test composition. The test compositions will comprise a BPI protein product, e.g. rBPl23, with or without known antibiotics and/or surfactants. Untreated infected specimens are used as a control. Armadillos from each treatment regimen are examined and biopsy specimens analyzed by procedures known in the art. M. leprae isolated from armadillos is assayed for metabolic activity. Differences between the appearance of lesions, differences in M. leprae bacterial concentrations, and differences in the metabolic activity of M. leprae isolates in treated versus control specimens are indicative of the bacteriostatic or bactericidal efficacy of a given BPI treatment regimen. EXAMPLE 11 The following experiment is conducted to determine the level of decontamination of a fluid containing LAM that can be achieved by treatment with a BPI protein product. Whole human blood, plasma, blood serum or the like is passed through a column containing a matrix, to which a BPI protein product is bound. Such matrix may be constructed by any means known to those skilled in the art. LAM in the fluid complexes with the BPI protein product affixed to the matrix as the fluid is passed through the column. The absence of LAM in the fluid eluted from the column demonstrates the effectiveness of a BPI protein product at decontaminating a fluid containing LAM. Alternatively, monoclonal antibodies with binding specificity for a BPI protein product, such as the antibodies employed in Example 1, are affixed to the matrix. A sufficient amount of a BPI protein product is added to the mixture to bind any LAM present in the fluid. The fluid is purified by passing it through the column. The αBPI antibodies affixed to the column bind the LAM/BPI protein product complex in the fluid, and the fluid eluted from the column is analyzed for the presence or absence of LAM contamination. Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description of the presently preferred embodiments thereof. For example, while the above illustrative examples principally address studies predictive of antibacterial effects in the context M.tuberculosis and M. leprae, model studies of infection with, e.g., M.avium [see, e.g., Brown et al, Antimicrob. Agents and Therapy, 37(3): 398-402(1993)] are also expected to reveal effectiveness of BPI protein product therapies. Consequently, the only limitations which should be placed upon the scope of the present invention are those which appear in the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: XOMA Corporation (ii) TITLE OF INVENTION: Treatment of Mycobacterial Diseases by Administration of Bactericidal/Permeability-Increasing Protein Product (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Marshall, O'Toole, Gerstein, Murray & Borun (B) STREET: 6300 Sears Tower, 233 South Wac er Drive (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: USA (F) ZIP: 60606-6402 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version 1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: ι (C) CLASSIFICATION: (viii) ATTORNEY INFORMATION: (A) NAME: Borun, Michael F. (B) REGISTRATION NUMBER: 25,447 (C) REFERENCE/DOCKET NUMBER: 31293 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 312/474-6300 (B) TELEFAX: 312/474-0448 (C) TELEX: 25-3856 (2) INFORMATION FOR SEQ ID NO:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1813 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 31..1491 (ix) FEATURE: (A) NAME/KEY: mat_peptide (B) LOCATION: 124..1491 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: CAGGCCTTGA GGTTTTGGCA GCTCTGGAGG ATG AGA GAG AAC ATG GCC AGG GGC 54 Met Arg Glu Asn Met Ala Arg Gly -31 -30 -25 CCT TGC AAC GCG CCG AGA TGG GTG TCC CTG ATG GTG CTC GTC GCC ATA 102 Pro Cys Asn Ala Pro Arg Trp Val Ser Leu Met Val Leu Val Ala He -20 -15 -10 GGC ACC GCC GTG ACA GCG GCC GTC AAC CCT GGC GTC GTG GTC AGG ATC 150 Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gly Val Val Val Arg He -5 1 5 TCC CAG AAG GGC CTG GAC TAC GCC AGC CAG CAG GGG ACG GCC GCT CTG 198 Ser Gin Lys Gly Leu Asp Tyr Ala Ser Gin Gin Gly Thr Ala Ala Leu 10 15 20 25 CAG AAG GAG CTG AAG AGG ATC AAG ATT CCT GAC TAC TCA GAC AGC TTT 246 Gin Lys Glu Leu Lys Arg He Lys He Pro Asp Tyr Ser Asp Ser Phe 30 35 40 AAG ATC AAG CAT CTT GGG AAG GGG CAT TAT AGC TTC TAC AGC ATG GAC 294 Lys He Lys His Leu Gly Lys Gly His Tyr Ser Phe Tyr Ser Met Asp 45 50 55 ATC CGT GAA TTC CAG CTT CCC AGT TCC CAG ATA AGC ATG GTG CCC AAT 342 He Arg Glu Phe Gin Leu Pro Ser Ser Gin He Ser Met Val Pro Asn 60 65 70 GTG GGC CTT AAG TTC TCC ATC AGC AAC GCC AAT ATC AAG ATC AGC GGG 390 Val Gly Leu Lys Phe Ser He Ser Asn Ala Asn He Lys He Ser Gly 75 80 85 AAA TGG AAG GCA CAA AAG AGA TTC TTA AAA ATG AGC GGC AAT TTT GAC 438 Lys Trp Lys Ala Gin Lys Arg Phe Leu Lys Met Ser Gly Asn Phe Asp 90 95 100 105 CTG AGC ATA GAA GGC ATG TCC ATT TCG GCT GAT CTG AAG CTG GGC AGT 486 Leu Ser He Glu Gly Met Ser He Ser Ala Asp Leu Lys Leu Gly Ser 110 115 120 AAC CCC ACG TCA GGC AAG CCC ACC ATC ACC TGC TCC AGC TGC AGC AGC 534 Asn Pro Thr Ser Gly Lys Pro Thr He Thr Cys Ser Ser Cys Ser Ser 125 130 135 CAC ATC AAC AGT GTC CAC GTG CAC ATC TCA AAG AGC AAA GTC GGG TGG 582 His He Asn Ser Val His Val His He Ser Lys Ser Lys Val Gly Trp 140 145 150 CTG ATC CAA CTC TTC CAC AAA AAA ATT GAG TCT GCG CTT CGA AAC AAG 630 Leu He Gin Leu Phe His Lys Lys He Glu Ser Ala Leu Arg Asn Lys 155 160 165 ATG AAC AGC CAG GTC TGC GAG AAA GTG ACC AAT TCT GTA TCC TCC AAG 678 Met Asn Ser Gin Val Cys Glu Lys Val Thr Asn Ser Val Ser Ser Lys 170 175 180 185 CTG CAA CCT TAT TTC CAG ACT CTG CCA GTA ATG ACC AAA ATA GAT TCT 726 Leu Gin Pro Tyr Phe Gin Thr Leu Pro Val Met Thr Lys He Asp Ser 190 195 200 GTG GCT GGA ATC AAC TAT GGT CTG GTG GCA CCT CCA GCA ACC ACG GCT 774 Val Ala Gly He Asn Tyr Gly Leu Val Ala Pro Pro Ala Thr Thr Ala 205 210 215 GAG ACC CTG GAT GTA CAG ATG AAG GGG GAG TTT TAC AGT GAG AAC CAC 822 Glu Thr Leu Asp Val Gin Met Lys Gly Glu Phe Tyr Ser Glu Asn His 220 225 230 CAC AAT CCA CCT CCC TTT GCT CCA CCA GTG ATG GAG TTT CCC GCT GCC 870 His Asn Pro Pro Pro Phe Ala Pro Pro Val Met Glu Phe Pro Ala Ala 235 240 245 CAT GAC CGC ATG GTA TAC CTG GGC CTC TCA GAC TAC TTC TTC AAC ACA 918 His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp Tyr Phe Phe Asn Thr 250 255 260 265 GCC GGG CTT GTA TAC CAA GAG GCT GGG GTC TTG AAG ATG ACC CTT AGA 966 Ala Gly Leu Val Tyr Gin Glu Ala Gly Val Leu Lys Met Thr Leu Arg 270 275 280 GAT GAC ATG ATT CCA AAG GAG TCC AAA TTT CGA CTG ACA ACC AAG TTC 1014 Asp Asp Met He Pro Lys Glu Ser Lys Phe Arg Leu Thr Thr Lys Phe 285 290 295 TTT GGA ACC TTC CTA CCT GAG GTG GCC AAG AAG TTT CCC AAC ATG AAG 1062 Phe Gly Thr Phe Leu Pro Glu Val Ala Lys Lys Phe Pro Asn Met Lys 300 305 310 ATA CAG ATC CAT GTC TCA GCC TCC ACC CCG CCA CAC CTG TCT GTG CAG 1110 He Gin He His Val Ser Ala Ser Thr Pro Pro His Leu Ser Val Gin 315 320 325 CCC ACC GGC CTT ACC TTC TAC CCT GCC GTG GAT GTC CAG GCC TTT GCC 1158 Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val Asp Val Gin Ala Phe Ala 330 335 340 345 GTC CTC CCC AAC TCC TCC CTG GCT TCC CTC TTC CTG ATT GGC ATG CAC 1206 Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu He Gly Met His 350 355 360 ACA ACT GGT TCC ATG GAG GTC AGC GCC GAG TCC AAC AGG CTT GTT GGA 1254 Thr Thr Gly Ser Met Glu Val Ser Ala Glu Ser Asn Arg Leu Val Gly 365 370 375 GAG CTC AAG CTG GAT AGG CTG CTC CTG GAA CTG AAG CAC TCA AAT ATT 1302 Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Leu Lys His Ser Asn He 380 385 390 GGC CCC TTC CCG GTT GAA TTG CTG CAG GAT ATC ATG AAC TAC ATT GTA 1350 Gly Pro Phe Pro Val Glu Leu Leu Gin Asp He Met Asn Tyr He Val 395 400 405 CCC ATT CTT GTG CTG CCC AGG GTT AAC GAG AAA CTA CAG AAA GGC TTC 1398 Pro He Leu Val Leu Pro Arg Val Asn Glu Lys Leu Gin Lys Gly Phe 410 415 420 425 CCT CTC CCG ACG CCG GCC AGA GTC CAG CTC TAC AAC GTA GTG CTT CAG 1446 Pro Leu Pro Thr Pro Ala Arg Val Gin Leu Tyr Asn Val Val Leu Gin 430 435 440 CCT CAC CAG AAC TTC CTG CTG TTC GGT GCA GAC GTT GTC TAT AAA 1491 Pro His Gin Asn Phe Leu Leu Phe Gly Ala Asp Val Val Tyr Lys 445 450 455 TGAAGGCACC AGGGGTGCCG GGGGCTGTCA GCCGCACCTG TTCCTGATGG GCTGTGGGGC 1551 ACCGGCTGCC TTTCCCCAGG GAATCCTCTC CAGATCTTAA CCAAGAGCCC CTTGCAAACT 1611 TCTTCGACTC AGATTCAGAA ATGATCTAAA CACGAGGAAA CATTATTCAT TGGAAAAGTG 1671 CATGGTGTGT ATTTTAGGGA TTATGAGCTT CTTTCAAGGG CTAAGGCTGC AGAGATATTT 1731 CCTCCAGGAA TCGTGTTTCA ATTGTAACCA AGAAATTTCC ATTTGTGCTT CATGAAAAAA 1791 AACTTCTGGT TTTTTTCATG TG 1813 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 487 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Arg Glu Asn Met Ala Arg Gly Pro Cys Asn Ala Pro Arg Trp Val -31 -30 -25 -20 Ser Leu Met Val Leu Val Ala He Gly Thr Ala Val Thr Ala Ala Val -15 -10 -5 1 Asn Pro Gly Val Val Val Arg He Ser Gin Lys Gly Leu Asp Tyr Ala 5 10 15 Ser Gin Gin Gly Thr Ala Ala Leu Gin Lys Glu Leu Lys Arg He Lys 20 25 30 He Pro Asp Tyr Ser Asp Ser Phe Lys He Lys His Leu Gly Lys Gly 35 40 45 His Tyr Ser Phe Tyr Ser Met Asp He Arg Glu Phe Gin Leu Pro Ser 50 55 60 65 Ser Gin He Ser Met Val Pro Asn Val Gly Leu Lys Phe Ser He Ser 70 75 80 Asn Ala Asn He Lys He Ser Gly Lys Trp Lys Ala Gin Lys Arg Phe 85 90 95 Leu Lys Met Ser Gly Asn Phe Asp Leu Ser He Glu Gly Met Ser He 100 105 110 Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr 115 120 125 He Thr Cys Ser Ser Cys Ser Ser His He Asn Ser Val His Val His 130 135 140 145 He Ser Lys Ser Lys Val Gly Trp Leu He Gin Leu Phe His Lys Lys 150 155 160 He Glu Ser Ala Leu Arg Asn Lys Met Asn Ser Gin Val Cys Glu Lys 165 170 175 Val Thr Asn Ser Val Ser Ser Lys Leu Gin Pro Tyr Phe Gin Thr Leu 180 185 190 Pro Val Met Thr Lys He Asp Ser Val Ala Gly He Asn Tyr Gly Leu 195 200 205 Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Leu Asp Val Gin Met Lys 210 215 220 225 Gly Glu Phe Tyr Ser Glu Asn His His Asn Pro Pro Pro Phe Ala Pro 230 235 240 Pro Val Met Glu Phe Pro Ala Ala His Asp Arg Met Val Tyr Leu Gly 245 250 255 Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Leu Val Tyr Gin Glu Ala 260 265 270 Gly Val Leu Lys Met Thr Leu Arg Asp Asp Met He Pro Lys Glu Ser 275 280 285 Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Thr Phe Leu Pro Glu Val 290 295 300 305 Ala Lys Lys Phe Pro Asn Met Lys He Gin He His Val Ser Ala Ser 310 315 320 Thr Pro Pro His Leu Ser Val Gin Pro Thr Gly Leu Thr Phe Tyr Pro 325 330 335 Ala Val Asp Val Gin Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala 340 345 350 Ser Leu Phe Leu He Gly Met His Thr Thr Gly Ser Met Glu Val Ser 355 360 365 Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Asp Arg Leu Leu 370 375 380 385 Leu Glu Leu Lys His Ser Asn He Gly Pro Phe Pro Val Glu Leu Leu 390 395 400 Gin Asp He Met Asn Tyr He Val Pro He Leu Val Leu Pro Arg Val 405 410 415 Asn Glu Lys Leu Gin Lys Gly Phe Pro Leu Pro Thr Pro Ala Arg Val 420 425 430 Gin Leu Tyr Asn Val Val Leu Gin Pro His Gin Asn Phe Leu Leu Phe 435 440 445 Gly Ala Asp Val Val Tyr Lys 450 455

Claims: I CLAIM: 1. A method of treating a subject suffering from infection with Mycobacteria which comprises administering to the subject a composition comprising a BPI protein product. 2. The method of claim 1 wherein the composition is administered orally. 3. The method of claim 1 wherein the composition is administered intravenously. 4. The method of claim 1 wherein the composition is administered as an aerosol. 5. The method of claim 1 wherein the BPI protein product is an 23-25 kD amino-terminal fragment of Bactericidal/permeability-increasing holoprotein. 6. The method of claim 1 for the treatment of infection with a Mycobacterium species bacterium selected from the group consisting of M.tuberculosis, M.leprae and M.avium 7. The method of claim 1 wherein the composition further comprises an antibiotic. 8. The method of claim 1 wherein the composition further comprises a surfactant. 9. A method of treating a subject suffering from the adverse physiological effects of the presence of lipoarabinomannan in circulation, said method comprising administering to the subject to the subject a composition comprising a BPI protein product. 10. The method of claim 9 wherein the adverse physiological effects comprise compromised immune response to microbes or tumor cells due to lipoarabinomannan-induced inhibition of macrophage activation by T- cell lymphokines. 11. The method of claim 9 wherein the adverse physiological effects comprise increased production of a cytokine by the subject. 12. The method of claim 9 wherein the composition is administered orally. 13. The method of claim 9 wherein the composition is administered intravenously. 14. The method of claim 9 wherein the composition is administered as an aerosol. 15. The method of claim 9 wherein the BPI protein product is a 23-25 kD amino-terminal fragment of Bactericidal/permeability-increasing protein. 16. The method of claim 9, wherein the composition further comprises a surfactant. 17. A method for decontaminating a fluid containing lipoarabinomannan said method comprising contacting the fluid with a BPI protein product under conditions such that lipoarabinomannan therein binds the BPI protein product and separating said bound materials from said fluid. 18. The method of claim 17, wherein the fluid is selected from the group consisting of blood, plasma, blood serum, and bone marrow. 19. The method of claim 17, wherein the fluid is selected from the group consisting of an isotonic solution, a pharmaceutical agent, and a cell culture reagent. 20. A BPI protein product for use in treating a subject suffering from infection with Mycobacteria. 21. A BPI protein product for use in treating a subject suffering from the adverse physiological effects of the presence of lipoarabinomannan in circulation. 22. A BPI protein product for use in decontaminating a fluid containing lipoarabinomannan. 23. Use of a BPI protein product for the manufacture of a medicament for treatment of Mycobacteria infection. 24. Use of a BPI protein product for the manufacture of a medicament for treatment of the adverse physiological effects of the presence of lipoarabinomannan in circulation. 25. A pharmaceutical composition for treatment of Mycobacteria infection comprising an effective amount of a BPI protein product. 26. A pharmaceutical composition for treatment of the adverse physiological effects of the presence of lipoarabinomannan in circulation comprising a BPI protein product.

Inventors: LAMBERT LEWIS H JR, LAMBERT, LEWIS, H., JR.
Applicants: XOMA CORP, XOMA CORPORATION
Application year: 1994
Main classification: A61K37
IPC codes: A61K38